In the past two decades mutations in the mitochondrial genome were found to be responsible for a significant burden of human disease. More recently mtDNA mutations were shown to accumulate over time and may be responsible for some phenotypes of aging. Whereas DNA transfections into the nuclear genome are commonplace and have yielded great insight into biology, no mitochondrial genome engineering methods have been published. Further complicating this methodological hurdle is the fact that multiple mitochondrial genomes can exist within a single mitochondrion and tens of hundreds of genomes within a single cell. What is required is a strategy that not only delivers DNA to mitochondria but also selectively removes and replaces the mitochondrial genomes in the cell. Gencia has developed Protofection(tm), a technology capable of delivering full-length mitochondrial DNA in vitro and in vivo, as well as a cloning method capable of producing full- length human mitochondrial DNA in a modified bacteriophage lambda vector and host. Through the use of protofection and a restriction-enzyme strategy, Gencia will prove the concept of fully replacing defective mtDNA as a therapy (or, Mitochondrial Genome Replacement Therapy - MGRT) for mitochondrial disease. In this Phase I SBIR, Gencia will develop MGRT for the treatment of a major cause of childhood blindness, LHON. MGRT will be used to treat a cybrid cell line derived from a LHON patient with wild-type cloned human mtDNA. This will result in a shift of the heteroplasmic ratios towards the healthy, wild-type engineered genome. The degree of correction of the LHON biochemical phenotype will be analyzed and correlated with the amount of engineered mtDNA delivered to cells. The specific proof-of-concept tasks are: (i) produce sufficient amounts of cGLP quality human and mouse cloned mtDNA as well as the protofection protein vector;(ii) introduce cloned human mtDNA expressing a restriction enzyme into a homoplasmic LHON cell line aiming for at least 90% content of delivered DNA, less than 10% cell death;and (iii) verify that this degree of replacement leads to measurable improvements in biochemical phenotypes. If successful, a Phase II SBIR will target testing of MGRT in animals to prove the ability to shift heteroplasmic ratios towards delivered mtDNA in vivo. While the goal of the project is to provide proof-of-concept of MGRT for LHON and to conduct important pre-clinical testing, insights and discoveries generated in this translational project should also validate MGRT as a platform technology for the treatment of other mitochondrial diseases. PUBLIC HEALTH RELEVANCE: The aims carried out under this proposal will show feasibility for MGRT (Mitochondrial Genome Replacement Therapy) as a treatment for Leber's Hereditary Optic Neuropathy (LHON), a form of blindness caused by mutations in mtDNA. In the 130 years since LHON was described, no effective treatments for the disease have been found. Generations of young adults still lose sight, usually permanently. Successful achievement of the aims will also provide a basis for treating other mitochondrial diseases. The potential impact on public health is significant. Although the incidence of most individual inherited mitochondrial diseases is low, taken as a group, they afflict an estimated 500,000 people in the US alone.